Antibiotics constitute one of the most effective classes of therapeutic agents developed over the last century; however, the sustained use of these compounds has led to widespread bacterial resistance, such as in methicillin-resistant Staphylococcus aureus (MRSA), necessitating the development of novel antibacterial compounds. Berninamycin A, from the thiazolyl peptide family, has shown activity against Gram-positive bacteria, including MRSA. Currently, the sequence of events comprising the biosynthesis of berninamycin is unknown as are the structural requirements for its formation in the producing organism. We will therefore: 1) reconstitute berninamycin in vitro from the pre-berninamycin peptide, utilizing the appropriate enzymes from the Streptomyces bernensis gene cluster and 2) investigate the substrate structural requirements for berninamycin synthesis in vivo, through genetic manipulation of S. bernensis. The information garnered from this study will increase our knowledge of how Nature constructs these architecturally unique heterocyclic molecules and may lead to the development of more potent medicines, especially antibiotics.